Studies On Construction And Expression Of Eucaryotic Recombinant Vector With HN Gene Of Goose Paramyxovirus

A new viral disease caused by goose paramyxovirus is an acute and violent infectious disease for geese, which characterized high mortality and result to a serious disserve in goose breeding industry.At present, there is not specific medicine to control GPMV in clinica. Vaccination is the effective means to prevent this disease. However, traditional live vaccine and inactive vaccine could not produce enough protective innmunity or not induce enough cytotoxic T lymphocytes(CTL) which is effective immunity cell and plays an important role in antivirus. With the development of molecular biology, how to prevent and control the disease with genetic engineering vaccine becomes an urgent problem.According to studies on GPMV, the enveloped virus has a negative-sense, single-stranded RNA genome of 15,186 nucleotides which codes for six proteins including nucleoprotein(N), phosphoprotein(P), matrix(M) protein, RNA-directed RNA polymerase(L), hemagglutinin-neuraminidase(HN) protein and fusion(F) protein from the 3' to 5'. Two interactive surface glycoproteins, the fusion(F) prteins and hemagglutinin-neuraminidase(HN) proteins play essential roles on GPMV attachment and fusion of cells during infection. Antibodies induced by the F or HN protein can neutralize the virus. Therefore, it is significant to construct an eucaryon expression vector with major protective antigen gene F or HN as DNA vaccine to prevent the disease.In this study, we designed a pair of specific primer according to the open reading fram(ORF) of HN gene of GPMV in GenBank. The virus were isolated from died goose with typical clinical symptoms, then cultured in 9 to 10-day-old embryonated SPF chicken eggs. After 24 hours, we attained the allantoid fluid that contains GPMV titer 2⁸ of hemagglutination. We extracted the virus RNA with TRIZOL according to the standard protocol, and amplified the HN fragment from GPMV strain JAU04.The HN gene was cloned into pMD18-T vector and transformed into E.coli JM109 to amplify. The recombinant plasmid with the HN gene fragment was sequenced(GenBank accession number EF141104). The result of sequence analysis showed that HN gene was composed of 1 716 bp and included a complete open reading frame which encoded a protein of 571 amino acids, including 5 protential asparagines-linked glycosylation sites and 12 conservative cysteines.Comparing the gene HN nucleotide and amino acids sequence with those from other goose paramyxoviruses revealed that homology of the nucleotide sequence was 81.9%-99.4% and amino acids sequence 88.4%-98.1%. In the end, HN gene was subcloned into pVAXI expression vector and transfected into Vero cells by means of Lipofectamine ^TM2000. Then the total RNA of the Vero cells were extracted and amplified. At the same time, HN gene was detected by RT-PCR and the HN protein was detected by directed fluorescence antibody. The result showed that a DNA fragment was found between 1 000-2 000 bp by RT-PCR, and the specific protein of HN expressed by pVAXI-HN which was detected with fluorescence antibodies. The nucleic acid vaccine pVAXI-HN with immunogenicity has been successfully constructed. And the pVAXI-HN can be expressed in eukaryocyte, which did the groundwork for the exploition of GPMV nucleic acid vector.